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Trophic effect of β-amyloid precursor protein on cerebral cortical neurons in culture
Vol. 181, No. 1, 1991
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Pages 265-271
November 27, 1991
TROPHIC EFFECT OF ~ -AMYLOID PRECURSOR PROTEIN ON CEREBRAL CORTICAL NEURONS IN CULTURE
Wataru Araki 1, Nobuya Kitaguchi2, Yasuo Tokushima 2, Kazuhiro Ishii 3, Hisashi Aratake 2, Shun Shimohama 1, Shigenobu Nakamura 4 and J u n Kimura 1
1Department of Neurology, Faculty of Medicine and 3Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan 2Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co. Ltd., Fuji-shi, Shizuoka 416, Japan 4Third Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan Received October 14, 1991
SUMMARY We investigated the effect of human ~-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons. ©1 9 9 1 A c a d . . . . P . . . . . I n c .
The amyloid B-protein is a major constituent of the senile plaque and vascular amyloid in Alzheimer's disease (AD) (1,2) and derived from a larger ~-amyloid precursor protein (APP) (3). At least three isoforms of APP (APP695, APP751 and APP770) have been identified, two of which (APP751 and APP770) contain an insert homologous to protease inhibitors of the Kunitz type (KPI) (4). APP mRNAs are expressed in almost every tissue, but the ratio of three isoforms differs among tissues. (5,6). The APPs have a characteristic structure of an integral membrane glycoprotein, with the B-protein region derived from portions of the extracellular and transmembrane domain (3). Many types of cell lines have been shown to secrete APPs lacking the cytoplasmic domain (7-9). Secreted APP possessing the KPI-domain appears to be identical to the protease nexin II which is secreted by fibroblasts (10,11).
265
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Recent reports using a cell culture system have indicated that APP m a y play a role in the regulation of the fibroblast growth (12) and in the modulation of cell adhesion (13-15). In addition, B-protein itself and a C-terminal fragment of APP containing the B-protein region have been reported to exert neurotoxic and neurotrophic effects on cultured neurons (16-19). However, little is known as to w h e t h e r or not secreted forms of APP have any effect on neuronal cells. In the present study, we investigated the effect of secreted APP on rat primary cerebral cortical neurons using APP species obtained from COS-1 cells transfected with cDNAs, which encode the N-terminal portions of APP770 and APP695.
MATERIALS AND METHODS Cell Culture The cerebral cortices of 17-day embryonic Wistar rats were dissected in Ca, Mgfree H a n k s ' balanced salt solution (HBSS), and i n c u b a t e d in 0.1% t r y p s i n containing 10 ~g/ml DNase I in Ca, Mg-free HBSS, for 5 min at 37°C. The tissues were washed with 1% BSA in Ca, Mg-free HBSS; and dissociated by pipetting in a serum-free m e d i u m (SFM), a modification of the N2 medium as described by Bottenstein and Sato (20). SFM consisted of 1:1 mixture of Dulbecco's modified medium (DMEM) and Ham's F12 supplemented with 0.67 ~g/ml vitamin BI~ 810 ~g/ml glucose, 73 ~g/ml glutamine, 50 ~g/ml gentamicin and the N2 supplements (20), at pH 7.4. The cell suspension was centrifuged at 1000 rpm for 5 min and resuspended in SFM. After filtration through a double layer of stainless steel mesh (50 ~m), cells were again centrifuged and resuspended in SFM. The cells were plated at a density of about 2 X 104 cells/cm 2 on a 24-well plate (Coming) coated with poly-D-lysine, and maintained at 37°C with 5% COz Production ~nd Purifi¢otion of APP APP667 and APP592, which correspond to APP770 and APP695 respectively, were produced by the method reported in a previous study (21) (Fig. 1A). APP667 and APP592 expression plasmids were constructed by the insertion of B a m H IHind III fragment from pUC18 into Bgl II-Hind III-digested pSVMT-APP770 and pSVMT-APP695 (4), respectively, to replace the C-terminal 104 a.a. (amino acids) of the parent APPs with a leucine residue. The plasmids were transfected into COS-1 cells by the method described previously (22). We purified the secreted APP667 from the conditioned medium of the transfected cells by HPLC on a MONO-Q column and the secreted APP592 from the conditioned medium by HPLC on a DEAE-5PW and Heparin-5PW. The purified proteins obtained were confirmed to be homogenous on SDS-PAGE (Fig. 1B). The molecular weights of APP592 and APP667 estimated by SDS-PAGE were about 95,000 and 100,000 respectively. Determination of APP Effect APP592 or APP667 was added to the medium at the time of plating (Day 0) at various concentrations. Duplicate wells were treated with each concentration of APP. The c u l t u r e s were m a i n t a i n e d for 3 days, a n d t h e n fixed in 4% paraformaldehyde and 0.12 M sucrose in PBS at 37°C for 30 min (18). The number of viable neurons and the length of the longest neurite were measured under phase contrast microscopy by the method described in Table 1. 266
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B
~taO ¢" n
n
KDa 200.0116.397.4-
A
Membrane
•
100KDa
•
95KDa
66.2--
APP695
,
APP592 APP770
NI
APP667
,
.j
:
f~=: ~ '
i
v/~
'.c =
'
i
42.7--
I I03 a a "~,
I
Fig. 1. (A) Schematic representation of APP695, APP592, APP770 a n d APP667. The closed box r e p r e s e n t s l~-protein, dotted box the K u n i t z - t y p e p r o t e a s e i n h i b i t o r domain a n d hatched box the additional 19 a.a. insert. (B) SDS-PAGE of APP667 a n d APP592. The purified APP667 (left) a n d APP592 (right) were electrophoresed i n 7.5% polyacrylamide gel c o n t a i n i n g 0.1% SDS a n d stained with Coomassie B r i l l i a n t Blue.
Table 1. The effects of APP592 a n d APP667 on n e u r o n a l survival a n d the length of the longest n e u r i t e of cultured cortical n e u r o n s APP concentration Neuronal survival Length of longest n e u r i t e (nM) (cells/ram2) ~m) APP592 APP667 APP592 APP667
Experiment 1 0 (Control) 1 10 40 100 200
35.7 ± 2.5 n.t. 40.4 ± 0.1 38.822.4 38.824.5 44.2 + 1.4 43.0 ± 4.8 51.6 + 3.6 51.0 ± 3.4 n.t. 40.8 + 4.0
91 + 4.5 n.t. 90 -+5.5 111±7.7 110±8.5 126 + 6.8 ** 126 + 6.5 ** 148 ± 7.6 ** 132 +_8.5 ** n.t. 139 + 7.3 **
7.6 ± 2.6 5.8± 2.0 6.1±3.3 28.0±3.8* 21.8±0" 48.4 ± 4.8 * 43.0 ± 1.4 ** 57.0 ± 4.6 * 55.4 ± 1.6 ** 37.0 ± 1.4 ** 38.3 ± 1.4 **
71 ± 3.1 70±3.0 77 ±4.3 107 ± 5.8 ** 134 ± 8.2 ** 144 ± 7.3 **
Experiment 2 0 (Control) 1 10 40 100 200
68_+3.5 73±4.3 105 ± 6.1 ** 135 ± 7.8 ** 142 + 8.5 **
P r i m a r y cerebral cortical n e u r o n s were cultured with or without APP592 or APP667 for 3 days, a n d fixed as described i n M a t e r i a l s a n d Methods. We c o u n t e d the n u m b e r of surviving cells with distinct n e u r i t e s i n 20 fields (0.25 m m 2 each) per well u n d e r p h a s e - c o n t r a s t microscopy, scoring 2 wells for each t r e a t m e n t . Each v a l u e for n e u r o n a l survival r e p r e s e n t s the m e a n cell n u m b e r + S.E.M. of duplicate wells. We e s t i m a t e d the l e n g t h of the longest n e u r i t e u s i n g a n ocular m i c r o m e t e r i n 30 isolated n e u r o n s i n 2 wells for each t r e a t m e n t . Each v a l u e for the n e u r i t e l e n g t h r e p r e s e n t s the m e a n + S.E.M. The r e s u l t s of two i n d e p e n d e n t e x p e r i m e n t s are presented, n.t.: not tested. *p < 0.05, **p < 0.01, by the Student's t test, compared to the control. 267
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RESULTS AND DISCUSSION The cells obtained in our culture exhibited neuronal profiles with several thin neurites (Fig. 2). They were further identified as neurons by immunostaining with antibodies against neurofilament protein and microtubule-associated protein 2 (data not shown). In addition, they did not stain positively for glial fibrillary acidic protein (GFAP) (data not shown). We tested the effect of APP on the neurons by adding highly purified APP (APP592 and APP667) to the medium. As shown in Fig. 2, both the survival of neurons and neurite outgrowth were enhanced by the addition of APP592 or APP667. We analyzed these effects q u a n t i t a t i v e l y by measuring the n u m b e r of surviving cells and the length of the longest neurite. Table 1 shows the results of two independent experiments in which various doses of APP were used. Neuronal survival was enhanced with increasing doses of APP, with optimal survival observed at 100 nM. There appeared to be no difference in survival-promoting effect between APP592 and APP667. The difference in cell survival b e t w e e n control cultures in the two experiments m a y be due to cellular damage caused during dissociation. Both types of APP also increased the length of the longest neurite in a dose-dependent manner, and the increase in the neurite length was significant at concentrations higher than 40 nM. Interestingly, slight decline in neuronal survival was observed at 200 nM of APP, whereas neurite o u t g r o w t h was still stimulated. This observation suggest a possibility t h a t neuronal survival and neurite formation are regulated independently. It was noted that the extent of the outgrowth of shorter neurites was not altered much by the addition of APP. The long neurite m a y correspond to an axon, and short n e u r i t e s dendrites, b u t f u r t h e r e x a m i n a t i o n will be n e c e s s a r y for precise characterization. In addition, there were no GFAP-positive glia in the culture even in the presence of APP, suggesting that the effects of APP in our study were not modified by glial cells. We have clearly shown t h a t secreted APP has a t r o p h i c effect on cultured cerebral cortical neurons in terms of neuronal survival and neurite extension. Since the APP species used in this study lack C-terminal 103 a.a. of the parent APPs, and both APP592 and APP667 caused essentially the same effects, we can conclude that (i) the extracellular domain of APP excluding the B-protein region has the trophic effect and (ii) the KPI-domain is irrelevant to this effect. In addition, we have observed that a 72 a.a. fragment of APP770 including the KPI268
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Fig. 2. The effects of APP592 a n d APP667 on c u l t u r e d cortical n e u r o n s . Phasecontrast micrographs of Day 3 control culture (A) a n d cultures t r e a t e d with 100 n M APP592 (B) or 100 nM APP667 (C). Bar = 50 ~ m . 269
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domain (APPI-72) (22) had no clear effect on these cultured neurons (data not shown). These results suggest that APP exerts its effect by a different mechanism from that of glia-derived nexin (GDN), in which the neurite-promoting effect may involve protease inhibition (23). The mechanism of the trophic effect of APP remains to be clarified, b u t at least two possible assumptions can be made. First, the APP effect might result from enhancement of cell adhesion. Schubert et al. (13) have reported that both APP695 and APP751 are able to stimulate adhesion of PC12 cells to substrata. Second, APP might interact with a specific cell-surface receptor, activating a second messenger system. It is consistent with this idea t h a t a similar trophic effect on cortical neurons is produced by basic fibroblast growth factor (bFGF) most likely by a receptor-mediated mechanism (24). The results of the p r e s e n t in vitro s t u d y suggest t h a t APP m a y have a neurotrophic effect in vivo. The APP is expressed not only in neuronal cells b u t also in glial cells in the brain (25). In addition, cerebrospinal fluid and brain tissue appear to contain secreted forms of APP as in the case of certain cultured cells (7,8,21,26). Thus, secretory forms of APP m a y exist in the brain, and have trophic activity for central nervous system neurons possibly by an autocrine mechanism. Whether there is any relationship between the neurotrophic activity of APP and the pathogenesis of AD remains to be investigated. In conclusion, our results have d e m o n s t r a t e d that secreted APP produces a trophic effect on primary cerebral cortical neurons, and strongly indicate that APP has important physiological activities in the central nervous system.
REFERENCES 1. 2.
3. 4. 5.
Glenner, G.G. and Wong, C.W. (1984) Biochem. Biophys. Res. Comm. 120, 885-890. M a s t e r s , C.L., S i m m s , G., W e i n m a n n , N.A., B e y r e u t h e r , K., M u l t h a u p , G. a n d McDonald, L.A. (1985) Proc. Natl. Acad. Sci. USA 82, 4245-4249. Kang, J., Lemaire, H.G., Unterbeck, A., Salbaum, J.M., M a s t e r s , C.L., Grzeschik, G.M., B e y r e u t h e r , K. and Muller-Hill, B. (1987) Nature 325, 733-736. K i t a g u c h i , H., T a k a h a s h i , Y., T o k u s h i m a , Y., Shiojiri, S. a n d Ito, H. (1988) Nature 331, 530-531. Neve, R.L., Finch, E.A. a n d D a w e s , L.R. (1988) N e u r o n 1, 669677. 270
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6. Tanaka, S., Shiojiri, S., Takahashi, Y., Kitaguchi, N., Ito, H., K a m e y a m a , M., Kimura, J., N a k a m u r a , S. and Ueda, K. (1989) Biochem. Biophys. Res. Comm. 165, 1406-1414. 7. Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L. and Beyreuther, IL (1989) Cell 57, 115-126. 8. Cole, G.M., Schubert, D. and Saitoh, T. (1990) In Molecular Biology and Genetics of Alzheimer's Disease (T. Miyatake, D.J. Selkoe, and Y. Ihara Eds.), pp113-122, Elsevier, Amsterdam. 9. Esch, F.S., Keim, P.S., Beattie, E.C., Blacher, R.W., Culwell, A., Oltersdorf, T., McClure, D. and Ward, P.J. (1990) Science 248, 1122-1124. 10. Oltersdorf, T., Fritz, L.C., S c h e n k , D.B., L i e b e r b u r g , I., Johnson-Wood, K.L., Beattie, E.C., Wand, P.J., Blancher, R.W., Dovey, H.F. and Sinha, S. (1989) Nature 341, 144-147. 11. Van N o s t r a n d , W.E., Wagner, S.L., Suzuki, M., Choi, B.H., Farrow, J.S., Geddes, J.W., Cotman, C.W. and Cunningham, D.D. (1989) Nature 341, 546-549. 12. Saitoh, T., Sundsmo, M., Roch, J.-M., Kimura, N., Cole, G., Schubert, D., Olsterdorf, T. and Schenk, D.B. (1989) Cell 58, 615-622. 13. Schubert, D., Jin, L.-W., Saitoh, T. and Cole, G. (1989) Neuron 3, 689-694. 14. Breen, K.C., Bruce, M. and Anderson, B.H. (1991) J. Neurosci. Res. 28, 90-100. 15. Chen, M. and Yankner, B.A. (1991) Neurosci. Lett. 125, 223-226. 16. Whitson, J.S., Selkoe, D.J. and Cotman, C.W. (1989) Science 243, 1488-1490. 17. Y a n k n e r , B.A., Dawes, L.R., Fisher, S., Villa-Komaroff, L., Oster-Granite, M.L. and Neve, R.L. (1989) Science 245, 417-420. 18. Yankner, B.A., Duffy, L.K. and Kirschner, D.A. (1990) Science 250, 279-282. 19. Roher, A.E., Bull, M.J., Bhave, S.V. and Wakade, A.R. (1991) Biochem. Biophys. Res. Comm. 174, 572-579. 20. Bottenstein, J.E. and Sato, G.H. (1979) Proc. Natl. Acad. Sci. USA 76, 514-517. 21. K i t a g u c h i , N., T o k u s h i m a , Y., Oishi, K., " T a k a h a s h i , Y., Shiojiri, S., N a k a m u r a , S., Tanaka, S., Kodaira, R. and Ito, H. (1990) Biochem. Biophys. Res. Comm. 166, 1453-1459. 22. Kitaguchi, N., Takahashi, Y., Oishi, K., Shiojiri, S., Tokushima, Y. Utsunomiya, T. and Ito, H. (1990) Biochim. Biophys. Acta 1038, 105-113. 23. Farmer, L., Sommer, J. and Monnard, D. (1990) Dev. Neurosci. 12, 73-80. 24. Morrison, R.S., Sharma, A., de Vellis, J. and Bradshaw, R.A. (1986) Proc. Natl. Acad. SCI. USA. 83, 7537-7541. 25. Selkoe, D.J. (1991) Neuron 6, 487-498. 26. Palmert, M.R., Berman-Podlisny, M., Witker, D.S., Oltersdorf, T., Younkin, L.H., Selkoe, D.J. and Younkin, S.G. (1989) Proc. Natl. Acad. Sci. USA. 86, 6338~342.
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Pages 265-271
November 27, 1991
TROPHIC EFFECT OF ~ -AMYLOID PRECURSOR PROTEIN ON CEREBRAL CORTICAL NEURONS IN CULTURE
Wataru Araki 1, Nobuya Kitaguchi2, Yasuo Tokushima 2, Kazuhiro Ishii 3, Hisashi Aratake 2, Shun Shimohama 1, Shigenobu Nakamura 4 and J u n Kimura 1
1Department of Neurology, Faculty of Medicine and 3Institute for Virus Research, Kyoto University, Sakyo-ku, Kyoto 606, Japan 2Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co. Ltd., Fuji-shi, Shizuoka 416, Japan 4Third Department of Internal Medicine, Hiroshima University School of Medicine, Hiroshima 734, Japan Received October 14, 1991
SUMMARY We investigated the effect of human ~-amyloid precursor protein (APP) on rat primary cerebral cortical neurons cultured in a serum-free medium. Two secretory APP species (APP667 and APP592) with and without the protease inhibitor domain were produced by COS-1 cells transfected with APP cDNAs, which encode the N-terminal portions of APP770 and APP695. Both highly purified APP species, when added to the medium, enhanced neuronal survival and neurite extension in a dose-dependent manner with a maximum effect at approximately 100 nM. These results suggest that secreted forms of APP have trophic activity for cerebral cortical neurons. ©1 9 9 1 A c a d . . . . P . . . . . I n c .
The amyloid B-protein is a major constituent of the senile plaque and vascular amyloid in Alzheimer's disease (AD) (1,2) and derived from a larger ~-amyloid precursor protein (APP) (3). At least three isoforms of APP (APP695, APP751 and APP770) have been identified, two of which (APP751 and APP770) contain an insert homologous to protease inhibitors of the Kunitz type (KPI) (4). APP mRNAs are expressed in almost every tissue, but the ratio of three isoforms differs among tissues. (5,6). The APPs have a characteristic structure of an integral membrane glycoprotein, with the B-protein region derived from portions of the extracellular and transmembrane domain (3). Many types of cell lines have been shown to secrete APPs lacking the cytoplasmic domain (7-9). Secreted APP possessing the KPI-domain appears to be identical to the protease nexin II which is secreted by fibroblasts (10,11).
265
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Vol. 181, No. 1, 1991
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Recent reports using a cell culture system have indicated that APP m a y play a role in the regulation of the fibroblast growth (12) and in the modulation of cell adhesion (13-15). In addition, B-protein itself and a C-terminal fragment of APP containing the B-protein region have been reported to exert neurotoxic and neurotrophic effects on cultured neurons (16-19). However, little is known as to w h e t h e r or not secreted forms of APP have any effect on neuronal cells. In the present study, we investigated the effect of secreted APP on rat primary cerebral cortical neurons using APP species obtained from COS-1 cells transfected with cDNAs, which encode the N-terminal portions of APP770 and APP695.
MATERIALS AND METHODS Cell Culture The cerebral cortices of 17-day embryonic Wistar rats were dissected in Ca, Mgfree H a n k s ' balanced salt solution (HBSS), and i n c u b a t e d in 0.1% t r y p s i n containing 10 ~g/ml DNase I in Ca, Mg-free HBSS, for 5 min at 37°C. The tissues were washed with 1% BSA in Ca, Mg-free HBSS; and dissociated by pipetting in a serum-free m e d i u m (SFM), a modification of the N2 medium as described by Bottenstein and Sato (20). SFM consisted of 1:1 mixture of Dulbecco's modified medium (DMEM) and Ham's F12 supplemented with 0.67 ~g/ml vitamin BI~ 810 ~g/ml glucose, 73 ~g/ml glutamine, 50 ~g/ml gentamicin and the N2 supplements (20), at pH 7.4. The cell suspension was centrifuged at 1000 rpm for 5 min and resuspended in SFM. After filtration through a double layer of stainless steel mesh (50 ~m), cells were again centrifuged and resuspended in SFM. The cells were plated at a density of about 2 X 104 cells/cm 2 on a 24-well plate (Coming) coated with poly-D-lysine, and maintained at 37°C with 5% COz Production ~nd Purifi¢otion of APP APP667 and APP592, which correspond to APP770 and APP695 respectively, were produced by the method reported in a previous study (21) (Fig. 1A). APP667 and APP592 expression plasmids were constructed by the insertion of B a m H IHind III fragment from pUC18 into Bgl II-Hind III-digested pSVMT-APP770 and pSVMT-APP695 (4), respectively, to replace the C-terminal 104 a.a. (amino acids) of the parent APPs with a leucine residue. The plasmids were transfected into COS-1 cells by the method described previously (22). We purified the secreted APP667 from the conditioned medium of the transfected cells by HPLC on a MONO-Q column and the secreted APP592 from the conditioned medium by HPLC on a DEAE-5PW and Heparin-5PW. The purified proteins obtained were confirmed to be homogenous on SDS-PAGE (Fig. 1B). The molecular weights of APP592 and APP667 estimated by SDS-PAGE were about 95,000 and 100,000 respectively. Determination of APP Effect APP592 or APP667 was added to the medium at the time of plating (Day 0) at various concentrations. Duplicate wells were treated with each concentration of APP. The c u l t u r e s were m a i n t a i n e d for 3 days, a n d t h e n fixed in 4% paraformaldehyde and 0.12 M sucrose in PBS at 37°C for 30 min (18). The number of viable neurons and the length of the longest neurite were measured under phase contrast microscopy by the method described in Table 1. 266
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B
~taO ¢" n
n
KDa 200.0116.397.4-
A
Membrane
•
100KDa
•
95KDa
66.2--
APP695
,
APP592 APP770
NI
APP667
,
.j
:
f~=: ~ '
i
v/~
'.c =
'
i
42.7--
I I03 a a "~,
I
Fig. 1. (A) Schematic representation of APP695, APP592, APP770 a n d APP667. The closed box r e p r e s e n t s l~-protein, dotted box the K u n i t z - t y p e p r o t e a s e i n h i b i t o r domain a n d hatched box the additional 19 a.a. insert. (B) SDS-PAGE of APP667 a n d APP592. The purified APP667 (left) a n d APP592 (right) were electrophoresed i n 7.5% polyacrylamide gel c o n t a i n i n g 0.1% SDS a n d stained with Coomassie B r i l l i a n t Blue.
Table 1. The effects of APP592 a n d APP667 on n e u r o n a l survival a n d the length of the longest n e u r i t e of cultured cortical n e u r o n s APP concentration Neuronal survival Length of longest n e u r i t e (nM) (cells/ram2) ~m) APP592 APP667 APP592 APP667
Experiment 1 0 (Control) 1 10 40 100 200
35.7 ± 2.5 n.t. 40.4 ± 0.1 38.822.4 38.824.5 44.2 + 1.4 43.0 ± 4.8 51.6 + 3.6 51.0 ± 3.4 n.t. 40.8 + 4.0
91 + 4.5 n.t. 90 -+5.5 111±7.7 110±8.5 126 + 6.8 ** 126 + 6.5 ** 148 ± 7.6 ** 132 +_8.5 ** n.t. 139 + 7.3 **
7.6 ± 2.6 5.8± 2.0 6.1±3.3 28.0±3.8* 21.8±0" 48.4 ± 4.8 * 43.0 ± 1.4 ** 57.0 ± 4.6 * 55.4 ± 1.6 ** 37.0 ± 1.4 ** 38.3 ± 1.4 **
71 ± 3.1 70±3.0 77 ±4.3 107 ± 5.8 ** 134 ± 8.2 ** 144 ± 7.3 **
Experiment 2 0 (Control) 1 10 40 100 200
68_+3.5 73±4.3 105 ± 6.1 ** 135 ± 7.8 ** 142 + 8.5 **
P r i m a r y cerebral cortical n e u r o n s were cultured with or without APP592 or APP667 for 3 days, a n d fixed as described i n M a t e r i a l s a n d Methods. We c o u n t e d the n u m b e r of surviving cells with distinct n e u r i t e s i n 20 fields (0.25 m m 2 each) per well u n d e r p h a s e - c o n t r a s t microscopy, scoring 2 wells for each t r e a t m e n t . Each v a l u e for n e u r o n a l survival r e p r e s e n t s the m e a n cell n u m b e r + S.E.M. of duplicate wells. We e s t i m a t e d the l e n g t h of the longest n e u r i t e u s i n g a n ocular m i c r o m e t e r i n 30 isolated n e u r o n s i n 2 wells for each t r e a t m e n t . Each v a l u e for the n e u r i t e l e n g t h r e p r e s e n t s the m e a n + S.E.M. The r e s u l t s of two i n d e p e n d e n t e x p e r i m e n t s are presented, n.t.: not tested. *p < 0.05, **p < 0.01, by the Student's t test, compared to the control. 267
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RESULTS AND DISCUSSION The cells obtained in our culture exhibited neuronal profiles with several thin neurites (Fig. 2). They were further identified as neurons by immunostaining with antibodies against neurofilament protein and microtubule-associated protein 2 (data not shown). In addition, they did not stain positively for glial fibrillary acidic protein (GFAP) (data not shown). We tested the effect of APP on the neurons by adding highly purified APP (APP592 and APP667) to the medium. As shown in Fig. 2, both the survival of neurons and neurite outgrowth were enhanced by the addition of APP592 or APP667. We analyzed these effects q u a n t i t a t i v e l y by measuring the n u m b e r of surviving cells and the length of the longest neurite. Table 1 shows the results of two independent experiments in which various doses of APP were used. Neuronal survival was enhanced with increasing doses of APP, with optimal survival observed at 100 nM. There appeared to be no difference in survival-promoting effect between APP592 and APP667. The difference in cell survival b e t w e e n control cultures in the two experiments m a y be due to cellular damage caused during dissociation. Both types of APP also increased the length of the longest neurite in a dose-dependent manner, and the increase in the neurite length was significant at concentrations higher than 40 nM. Interestingly, slight decline in neuronal survival was observed at 200 nM of APP, whereas neurite o u t g r o w t h was still stimulated. This observation suggest a possibility t h a t neuronal survival and neurite formation are regulated independently. It was noted that the extent of the outgrowth of shorter neurites was not altered much by the addition of APP. The long neurite m a y correspond to an axon, and short n e u r i t e s dendrites, b u t f u r t h e r e x a m i n a t i o n will be n e c e s s a r y for precise characterization. In addition, there were no GFAP-positive glia in the culture even in the presence of APP, suggesting that the effects of APP in our study were not modified by glial cells. We have clearly shown t h a t secreted APP has a t r o p h i c effect on cultured cerebral cortical neurons in terms of neuronal survival and neurite extension. Since the APP species used in this study lack C-terminal 103 a.a. of the parent APPs, and both APP592 and APP667 caused essentially the same effects, we can conclude that (i) the extracellular domain of APP excluding the B-protein region has the trophic effect and (ii) the KPI-domain is irrelevant to this effect. In addition, we have observed that a 72 a.a. fragment of APP770 including the KPI268
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Fig. 2. The effects of APP592 a n d APP667 on c u l t u r e d cortical n e u r o n s . Phasecontrast micrographs of Day 3 control culture (A) a n d cultures t r e a t e d with 100 n M APP592 (B) or 100 nM APP667 (C). Bar = 50 ~ m . 269
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domain (APPI-72) (22) had no clear effect on these cultured neurons (data not shown). These results suggest that APP exerts its effect by a different mechanism from that of glia-derived nexin (GDN), in which the neurite-promoting effect may involve protease inhibition (23). The mechanism of the trophic effect of APP remains to be clarified, b u t at least two possible assumptions can be made. First, the APP effect might result from enhancement of cell adhesion. Schubert et al. (13) have reported that both APP695 and APP751 are able to stimulate adhesion of PC12 cells to substrata. Second, APP might interact with a specific cell-surface receptor, activating a second messenger system. It is consistent with this idea t h a t a similar trophic effect on cortical neurons is produced by basic fibroblast growth factor (bFGF) most likely by a receptor-mediated mechanism (24). The results of the p r e s e n t in vitro s t u d y suggest t h a t APP m a y have a neurotrophic effect in vivo. The APP is expressed not only in neuronal cells b u t also in glial cells in the brain (25). In addition, cerebrospinal fluid and brain tissue appear to contain secreted forms of APP as in the case of certain cultured cells (7,8,21,26). Thus, secretory forms of APP m a y exist in the brain, and have trophic activity for central nervous system neurons possibly by an autocrine mechanism. Whether there is any relationship between the neurotrophic activity of APP and the pathogenesis of AD remains to be investigated. In conclusion, our results have d e m o n s t r a t e d that secreted APP produces a trophic effect on primary cerebral cortical neurons, and strongly indicate that APP has important physiological activities in the central nervous system.
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